1. Field of the Invention
The present invention relates to a method for changing the percentage of mutant mitochondrial genomic DNA in a cell. In addition, the present invention relates to a method for generating homoplasmic cells according to the above-described method.
2. Description of the Related Art
A eukaryotic cell comprises several hundreds of to several thousands of copies of mitochondrial DNA (hereinafter referred to as “mtDNA”) in mitochondria thereof. Wild-type human mtDNA originally has a single genotype, namely, a single nucleotide sequence, in individual cells that constitute the whole body. This state is referred to as homoplasmy. To date, mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), chronic progressive external ophthalmoplegia (CPEO), myoclonic epilepsy with ragged-red fibers (MERRF)=Fukuhara disease, Leigh's encephalopathy, hypertrophic cardiomyopathy caused by mitochondrial DNA abnormity, Leber's disease, and Pearson syndrome have been known. There is no radical treatment for all these diseases. It has been known that, with regard to a majority of these mitochondrial diseases, mutant mtDNA coexists with wild-type mtDNA in a cell (this state is referred to as heteroplasmy), and that mitochondrial disease may be developed, when the percentage of such mutant mtDNA in the cell is high.
A person having mutant mtDNA was usually no symptom at birth. However, if the percentage of mutant mtDNA exceeds a certain threshold, clinical symptoms may be expressed. For example, it has been reported that most MELAS patients and some cardiomyopathy patients have a large mtDNA deletion of the nucleotide at position 3243, that most MERRF patients have a large mtDNA deletion of the nucleotide at position 8344, and that CPEO and Pearson syndrome patients also have a large deletion of the mtDNA thereof (Wallace, Annu Rev Genet., 39, 359-407, 2005). Moreover, it has been clarified that mutant mtDNA involving A3243G substitution may cause diabetes.
The aforementioned diseases caused by mutant mtDNA may all develop severe symptoms. However, at present, there have been no effective treatment. Thus, effective treatments have been strongly desired to be established as soon as possible.
If a heteroplasmic cell comprising both mutant mtDNA and wild-type mtDNA were converted to a homoplasmic cell having only normal mtDNA, it could provide an important key for developing the treatment of the mitochondrial diseases. To date, the present inventors have already revealed that only a moderate level of oxidative stress induces a special type of mtDNA replication (rolling circle DNA replication) (Ling and Shibata EMBO J., 21, 4730-4740, 2002; Ling et al., Mol. Cell. Biol., 27, 1133-1145, 2007), which is induced by the same mechanism as a homologous DNA recombination initiation mechanism in a budding yeast (Saccharomyces cerevisiae) (Hori et al., Nucleic Acids Res., 37, 749-761, 2009); and that homoplasmic cells can be generated from heteroplasmic cells by this rolling circle mtDNA replication (Ling and Shibata, Mol. Biol. Cell, 15, 310-322, 2004; Shibata and Ling, Mitochondrion, 7, 17-23, 2007).
On the other hand, it has been reported that mtDNA replication in mammalian cells including human cells is θ type replication (Kirschner et al., Proc. Natl. Acad. Sci. U.S.A., 60, 1466-1472, 1968). With regard to such mtDNA replication in mammalian cells including human cells, it has also been reported that a T4 phage type replication, in which recombination occurs together with replication, may be carried out (Pohjoismaki et al., J. Biol. Chem., 284, 21446-21457, 2009). At present, the details of such mechanisms have been in a state of chaos, and thus, have not yet been clarified. However, currently, it has been generally believed that rolling circle mtDNA replication is not a main mechanism (Bogenhangen and Clayton, Trends Biochem. Sci., 28, 357-360, 2003; Bogenhangen and Clayton, Trends Biochem. Sci., 28, 404-405, 2003).
In general, cells derived from a healthy human individual comprise wild-type mtDNA in a homoplasmic state. It has been reported that mtDNA deletions are present in egg cells collected from normal women (Chen et al., Am J Hum Genet, 57, 239 247, 1995). If this report is correct, it is considered that a fertilized egg can be converted to homoplasmic cells during the process of development and differentiation thereof. However, a mechanism for converting a fertilized egg to homoplasmic cells is totally unknown. Furthermore, as stated above, a mechanism for replicating mtDNA in mammalian cells including human cells differs from that for budding yeasts. Accordingly, it is not easy to predict a mechanism for converting mtDNA in mammalian cells to a homoplasmic state based on the mechanism for converting mtDNA in budding yeasts to a homoplasmic state.
Hence, in order to develop a method of medical treatment for mitochondrial diseases, it needs to promote further studies regarding the mechanisms for a homoplasmic state.